TYROSINE PHOSPHORYLATION AND [CA2-CELLS - IMPLICATIONS FOR CEREBRAL VASOSPASM(](I) ELEVATION INDUCED BY HEMOLYSATE IN BOVINE ENDOTHELIAL)

Endothelial cells are affected in the cerebral vasospasm that occurs a t the time of erthyrocyte lysis in a subarachnoid clot A red blood cel l lysate was added to bovine pulmonary artery endothelial cells in vit ro to determine whether hemolysate can trigger tyrosine kinase mediate d cell signalling and if so, whether this signal is independent of the elevation of intracellular free calcium levels, [Ca2+](i), induced by hemolysate. Hemolysate was found by Western blotting to induce a dose dependent increase in the level of tyrosine phosphorylation of two pr oteins, approximately 60 and 110 kD, that was maximal between 1 and 2 min. The biphasic increase in [Ca2+](i) induced by hemolysate consists of a peak complete within 1 min which is the result of release of int racellular calcium stores and a plateau phase due to an influx of extr acellular Ca2+. Addition of hemolysate to cells in the presence of EGT A indicated that an extracellular Ca2+ influx is not required for the increases in tyrosine phosphorylation. Release of intracellular Ca2+ s tores by thapsigargin, a Ca2+-ATPase inhibitor, was, however, found to increase the phosphotyrosine content of the same 60 and 110 kD protei ns. Endothelial cells pretreated with tyrosine kinase inhibitors, tyrp hostin 25 or genistein, before exposure to hemolysate blocked the plat eau phase of the [Ca2+](i) response indicating that tyrosine kinase ac tivity is required for the influx. Ca2+ and phosphotyrosine mediated c ell signalling induced by hemolysate in endothelial cells may be activ ated by a single component but represent distinct targets for possible control of the cerebral vasospasm response.